Enzyme substrates with naphthylamines as chromogenic groups linked to other amino acids have been described in the literature for the determination of transferases and proteases, such as .gamma.-glutamyl transpeptidase, lecuine aminopeptidase, oxytocinase, and trypsin. Orlowski et al, Clin. Chim, Acta, 7:755-760 (1962), and references cited therein. The determination of transferase and protease activity in human serum, urine, and tissues may have diagnostic significance; for example, the assay of .gamma.-glutamyl transpeptidase activity in human serum may be useful in the differential diagnosis of liver diseases, because the enzyme activity is particularly high in obstructive jaundice and liver cancer while lower activities are observed in viral hepatitis and liver cirrhosis. Orlowski et al, supra. See also Rosalki et al, Ann. Clin. Biochem. 7:143 (1970). The majority of studies with respect to .gamma.-glutamyl transpeptidase determinations have been carried out using naphthylamines in formulating the substrates and, unfortunately, the products (i.e., naphthylamines) are both toxic and carcinogenic, presenting undesirable risks for general laboratory use.
Many of the enzyme assays commonly performed in clinical laboratories are NADH linked; that is, they involve a series of reactions which ultimately result in the reduction of nicotinamide adenine dinucleotide (NAD) to its reduced form, NADH. The NADH is then detected spectrophotometrically or fluorometrically. The more recent fluorometric procedures have the characteristic advantages of simplicity, speed, and economy, and often have the further advantage of greater sensitivity. Typically, a fluorometric NADH-linked test involves the use of a filter fluorometer which directs ultraviolet light at a wavelength of about 340 nm against the surface of the sample and which measures the fluorescence, or rate of change of fluorescence at an emission wavelength of about 465 nm.
Other references illustrating the state of the prior art are U.S. Pat. Nos. 3,979,447, 3,862,011, 3,773,626, 3,591,458, 3,878,048, 3,892,631, and Wildes et al, J. Am. Chem. Soc., 95:8, 2610 (1973), and Bayley et al, Eur. J. Biochem. 56 (2), 455-65 (1975).